重组人抗狂犬病毒抗体的实验研究

发布时间：2018-02-09 20:49

本文关键词： 载体构建 scFv-Fc 抗体表达文库筛选大规模发酵全分子抗体毕赤酵母　出处：《吉林大学》2007年博士论文　论文类型：学位论文

【摘要】： 本研究利用基因重组技术构建了可在真核表达系统毕赤酵母中稳定有效表达人小分子抗体scFv-Fc的表达载体pPICZα/scFv-Fc,建立了一套可表达筛选人小分子抗体scFv-Fc的真核表达体系。应用该表达体系构建人抗狂犬病毒小分子抗体scFv-Fc的表达文库,利用抗体的抗原特异结合活性从中筛选人抗狂犬病毒小分子抗体scFv-Fc,结果获得了新的具有狂犬病毒抗原结合活性的抗体可变区序列,其中克隆RS3(轻链可变区为κ链)和RS9(轻链可变区为λ链)具有较好的scFv-Fc小分子抗体表达及狂犬病毒抗原结合活性。进而在80L发酵条件下,对毕赤酵母工程菌RS3表达条件进行了优化,并建立一种适合于大规模纯化人抗狂犬病毒小分子抗体scFv-Fc的方法。另外,我们应用基因重组技术,将筛选获得的抗狂犬病毒小分子抗体RS3的重链和轻链可变区基因重组到本实验室已构建的完整人重链及轻链表达载体pPICZαCH及pPICZαCκ,利用分步整合法转化X33酵母菌对抗狂犬病毒全分子抗体进行分泌表达,制备具狂犬病毒抗原结合活性的完整人抗体,鉴定重组抗体的生物学活性。结果表明,在毕赤酵母菌中可经甲醇诱导产生具抗原结合活性的完整分泌型抗体。本实验的创新之处在于:1)利用毕赤酵母构建了人小分子抗体scFv-Fc的表达筛选体系并获得了新的具有狂犬病毒抗原结合活性的抗体可变区序列,国内外尚未见报道;2)建立了大规模发酵和纯化人小分子抗体scFv-Fc的方法,国内尚未见报道;3)利用分步整合法电转化X33酵母菌,制备得到具狂犬病毒抗原结合活性的完整人抗体。
[Abstract]:In this study, the expression vector pPICZ 伪 -scFv-Fc, which can express human small molecule antibody scFv-Fc stably and effectively in Pichia pastoris, was constructed by gene recombination technique, and a set of eukaryotic expression system was established to express and screen human small molecule antibody scFv-Fc. The expression library of human anti-rabies virus small molecule antibody scFv-Fc was constructed by using this expression system. Using the antigen-specific binding activity of antibodies to human rabies virus small molecule antibody scFv-Fc, we obtained a new variable region of antibody with rabies virus antigen-binding activity. Among them, RS3 (light chain variable region is 魏 chain) and RS9 (light chain variable region is 位 chain) have good expression of scFv-Fc small molecule antibody and rabies virus antigen binding activity. The RS3 expression conditions of Pichia pastoris were optimized and a method was established for the large-scale purification of human anti-rabies virus small molecule antibody (scFv-Fc). In addition, we use gene recombination technology, The genes of heavy chain and light chain variable region of anti-rabies virus small molecule antibody RS3 were recombined into the complete human heavy chain and light chain expression vectors pPICZ 伪 Ch and pPICZ 伪 C 魏. The X33 yeast was transformed into X33 yeast by stepwise integration. The whole molecular antibody of canine virus is secreted and expressed. A complete human antibody with rabies virus antigen-binding activity was prepared and the biological activity of the recombinant antibody was identified. In Pichia pastoris, complete secretory antibodies with antigen-binding activity can be induced by methanol. The innovation of this experiment is that we constructed a screening system for the expression of human small molecule antibody scFv-Fc using Pichia pastoris and obtained a new one. A variable region of antibodies with rabies virus antigen-binding activity, The method of large-scale fermentation and purification of human small molecule antibody (scFv-Fc) has not been reported at home and abroad, and no report has been reported in China (3) the whole human antibody with rabies virus antigen-binding activity has been prepared by using stepwise integration method to electrotransform X33 yeast.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392

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